Chem. Res. Toxicol. 1996,8, 379-388
379
Reaction of Singlet Oxygen with 2’-Deoxyguanosineand DNA. Isolation and Characterization of the Main Oxidation Products Jean-Luc Ravanatt and Jean Cadet* CEAlDkpartement de Recherche Fondamentale sur la Matihre Condensee ISESAMILAN, F-38054 Grenoble Cedex 9, France Received October 5, 1994@
The reaction of singlet molecular oxygen with 2’-deoxyguanosine and DNA was studied. Emphasis was placed on the identification and characterization of the main methylene blue mediated type 11(singlet oxygen) oxidation products of 2’-deoxyguanosine and its corresponding 3‘,5‘-di-O-acetylated derivative. Two major oxidation products of 2‘-deoxyguanosine were isolated and characterized by mass spectrometry analysis and extensive l H and 1 3 C NMR measurements as the two 4R* and 4S* diastereomers of 4,8-dihydr0-4-hydroxy-8-0~0-2’deoxyguanosine. The addition of ‘ 0 2 was also found to occur to the base moiety of the corresponding 3’,5’-di-O-acetylated derivative. Methylene blue mediated photosensitization of 2’-deoxyguanosine led also to the production of 7,8-dihydro-8-0~0-2‘-deoxyguanosine, but in a relatively lower yield with respect to the two above diastereomers. The participation of singlet oxygen in the mechanism of formation of these oxidation products was confirmed. A reasonable mechanism involving the transient formation of an unstable endoperoxide produced through a Diels-Alder 1,4-cycloaddition of singlet oxygen to the purine ring is suggested. Quantitative analysis allowed us to demonstrate that the two diastereomers of 4,8-dihydro-4-hydroxy-8oxo-2’-deoxyguanosine are the main singlet oxygen oxidation products of the guanine moiety within nucleosides, whereas 7,8-dihydro-8-oxoguanine was found to be the major l o 2 oxidation product of guanine in double-stranded DNA.
Introduction The genotoxicity and the mutagenicity of l02, as the result of DNA oxidation, have been determined (for recent reviews see refs 1-3). A major source of lo2 is photosensitization reactions (4-6). Generation of ‘ 0 2 through energy transfer from an excited triplet-state sensitizer to ground-state molecular oxygen is defined as a type I1 photosensitization mechanism (7,8). Tyrrell et al. have suggested the involvement of singlet oxygen in the induction of the human heme oxygenase gene and in the inactivation of cultured human fibroblasts by UVA irradiation (9). Bezman et al. (10) have also shown the participation of ‘ 0 2 in the inactivation of Escherichia coli by rose bengal photosensitization. Singlet oxygen was also found to be able to induce the loss of biological activity of bacteria (10)and plasmids (11).It is important to note that lo2 may also be produced by other chemical and biochemical systems. Among others, we may cite the decomposition of dioxetanes (121, the dismutation of superoxide radical (13), and also enzymatic processes involving oxidases (14,151. Because of its relatively long lifetime, lo2 may diffuse toward potential targets and reacts like a strong electrophile with biomolecules that contain regions of high electron density. Therefore, cellular targets may include DNA (21, ribosomes (161, mitochondria (171, and cellular membranes (18). Among the nucleosides, lo2 has been shown to react preferentially with 2‘-deoxyguanosine (dGuo)’ (19-21 ), leading to complex mixtures of oxidation products which were only partly characterized (22,231. Exposure of DNA to *To whom correspondence and reprint requests should be addressed. Telephone (33)-76-88-49-87; Fax (33)-76-88-50-90. Present address: Nestle Research Centre, P.O. Box 44, CH-1000 Lausanne 26,Switzerland. Abstract published in Advance ACS Abstracts, March 1, 1995. +
@
0893-228x/95/2708-0379$09.0Ql0
led to base damage and strand breaks, both of which specificallyoccur at guanine residues (for reviews see refs 24-26). 7,8-Dihydro-8-0~0-2’-deoxyguanosine (8-oxodGuo) is the only lo2 mediated guanine modification that has been detected in DNA (27, 28). Several assays, including HPLC-electrochemical detection (HPLC-EC) (291, 32P-postlabeling(301, and also gas chromatographymass spectrometry (GC/MS) (31,321, are now available to monitor the formation of 8-oxodGuo, either as the nucleoside or as the free base, in both isolated and cellular DNA. In addition, a monoclonal antibody column is now available for the prepurification by affinity chromatography of 8-oxodGuo in urine (33). In the present work we report the results of the study on the type I1 (singlet oxygen) photosensitization reaction of 2’-deoxyguanosine and DNA. The main objective of the work was to provide a detailed characterization of the singlet oxygen mediated oxidation products of dGuo (1) and its corresponding 3’,5’-di-O-acetylatedderivative (la), whose structures were described in preliminary communications (34, 35).
Experimental Procedures Chemicals. Caution: Because of its toxicity, hydrogen fluoride (Aldrich Chemie, Milwaukee, WZ) should be handled with care, and reactions have to be carried out in nonglass flasks such as polypropylene tubes (Eppendorf R). Methylene blue and dGuo were purchased from Sigma Chemical Co. (St. Louis, MO) Abbreviations: 4-OH-8-oxodGu0,4,8-dihydro-4-hydroxyy-8-oxo-2’deoxyguanosine; 4-OH-8-oxoGua,4,8-dihydro-4-hydroxyy-8-oxoguanine; 8-oxodGu0, 7,8-dihydro-8-0~0-2’-deoxyguanosine; 8-oxoGua, 7,8-dihydro-8-oxoguanine; dGuo, 2’-deoxyguanosine; di-0-Ac-dGuo, 3’,5’-di-0acetyl-2’-deoxyguanosine;FAB/MS, fast atom bombardment mass spectrometry; HFPyr, hydrogen fluoride stabilized in pyridine; HPLCEC, high performance liquid chromatography-electrochemical detection; TSP, 3-trimethylsilyl(2,2,3,3-2H4)propionate.
0 1995 American Chemical Society
380 Chem. Res. Toxicol., Vol. 8, No. 3, 1995 and were used without further purification. 7,8-Dihydro-8oxoguanine (8-oxoGua) was obtained from Chemical Dynamics (South Plainfield, NJ). HPLC grade acetonitrile and ammonium formate were obtained from Carlo Erba (Farmitalia Carlo Erba, Milan, Italy) and Kodak (Eastman Kodak Co., Rochester, NY) respectively. Disulfonated phthalocyanine complexed with zinc which was a gift of Dr. J. E. van Lier (University of Sherbrooke, Sherbrooke, QuBbec) was prepared as described by Langlois et al. (36). Irradiation solutions containing Fe2+ were prepared by appropriate dilution of FeS04,(N&)zS04,6H20 purchased from Prolabo (Paris, France) to a 0.2 mM final concentration. A clean source of singlet oxygen was provided by the thermal decomposition of the endoperoxide of 3,3’-(1,4-naphthylidene)dipropionate (37). l s 0 z was obtained from Eurisotop (Paris, France). Irradiation System. The visible light was generated by a 100 W tungsten lamp, filtered with a 590 nm cutoff filter no. 23A (Eastman Kodak Co., Rochester, NY). During photolysis, the solution, placed a t a distance of 20 cm from the irradiation source, was kept saturated with oxygen by continuous air bubbling and maintained a t room temperature by circulating water. For isotopic labeling experiments, oxygen was removed from the irradiation solution under vacuum, and ‘ * 0 2 was introduced prior t o irradiation in a close flask. Spectrometer Analysis. W absorption spectra were recorded in H2O with a spectrophotometer DU 8B (Beckman, Fillerton, CAI. Circular dischroism spectra (for H2O solution) were obtained by using a Model I11 Roussel-Jouan dichrograph (Jobin-Yvon,Paris, France). 400.13 MHz IH NMR spectra were recorded on a Bruker AM-400 spectrometer a t 300 K either in 400 pL of 99.8% 2Hz0 (Merck, Darmstad, Germany) or in 400 pL of (C2H3)2S0(Merck, Darmstad, Germany). 3-(Trimethyl~ilyl)(2,2,3,3-~H4)propionate (TSP) was used as the internal standard (TSP = 0.00) in 2H20, whereas tetramethylsilane (TMS) was utilized for the calibration of the spectra in (C2H3)2SO. Assignments were checked by homonuclear double- or triple-resonance experiments. H2’ and H2” proton signals were further assigned on the basis of coupling constant arguments (38, 39). In order to obtain accurate coupling constants, the spectra were simulated using the Bruker PANIC program. 50.13 MHz 13CNMR spectra were obtained in (CZH3)2SOat 300 K with a Bruker AM-200 spectrometer. Long range heteronuclear scalar correlations were established by using the “INVDRSLP. A U Bruker program on the AM-400 spectrometer. Fast atom bombardment mass spectra (FAB/MS) (thioglycerol matrix, 35-keV Cs atoms) were obtained in the positive ion mode on a VG ZAB 2-EQ instrument (Fisons-V. G., Manchester, U.K.). HPLC Analysis. Several HPLC apparatus were used for both analytical and preparative separations. Large scale purification of synthesized di-O-Ac-dGuo (la) was achieved by using a Model 500 Waters HPLC preparative pumping system (Millipore, Mildford, MA) equipped with a differential refractometer and a P r e p p a c k 500 (500 x 50 mm id.) octadecylsilyl silica gel column (Millipore). The second system, used for semipreparative and analytical purifications of nucleoside photooxidation products, consisted of two Model 302 Gilson pumps (Middleton, WI) equipped with a Sil-SA Shimadzu automatic injector (Touzart & Matignon, Paris, France), a dynamic mixer Model 811 (Gilson), and a L-4000 spectrophotometer (Hitachi, Tokyo, Japan). The pumps were connected to an Apple I1 microcomputer that controled the eluent composition and the flow rate, usually set at 1 and 2 m u m i n for analytical and semipreparative separations, respectively. HPLC elution profiles were recorded and the peaks of interest were integrated by using a Model 621 Data Master (Gilson) interfaced with the microcomputer through the HPLC system manager software Model 704 (Gilson). Quantitation was achieved by using a n external calibration procedure. The HPLC-EC detection system consisted of a LKJ3 pump Model 2150 (Pharmacia LKB Biotechnology, Uppsala, Sweden) equipped with a Waters automatic injector Wisp 710B (Millipore) and a Model l l l B W detector (Gilson). The electrochemical detection was accomplished by amperometry using a Model LC-4B/LC-l7A(T)apparatus (Bio-
Ravanat and Cadet analytical Systems, West Lafayette, IN) set a t 0.5 nA. The amperometric detection of 8-oxoGua and its related nucleoside was achieved by using two glassy-carbon electrodes in parallel that were set a t a potential of f 6 5 0 mV (40). Analytical reverse phase columns (250 x 4.6 mm i d . ) for electrochemical detection were home-packed with 10 pm Nucleosil octadecylsilyl silica gel (Macherey Nagel, Duren, Germany). The analytical (250 x 4.6 mm i.d.) and semipreparative (250 x 6.2 mm i.d.) amino substituted silica gel (mean particle 5 pm) Hypersil NH2 columns were purchased from Interchim (Montluqon, France). For dose curve response studies, aliquots (200 pL) were periodically removed from the irradiated solutions, in order to monitor the formation of photoproducts by HPLC. For this purpose, 20 pL were injected onto the reverse phase column and 8-oxodGuo (4) was quantified by using the HPLC-EC assay, as previously described by Berger et al. (40). The formation of 4R* and 45* diastereomers 2 and 3 was monitored by introducing 50 pL of the photolyzed solution of 1 onto the analytical NH2 column, using a mixture of acetonitrile and 25 mM ammonium formate (80:20) as the mobile phase a t a flow rate of 1m u m i n (41). The 4R* and 4S* diastereomers 2 and 3 were detected a t 230 nm and quantified by external calibration. Chemical Synthesis. 8-OxodGuo (4) was prepared by hydrogenation of 8-(benzoyloxy)-2’-deoxyguanosineas previously described by Lin et al. (42). Di-O-Ac-dGuo (la)was synthesized by acetylation of 2’-deoxyguanosine (1). Typically, 2.67 g (10 mM) of dGuo (I) was dissolved in 17.5 mL of tetraethylammonium hydroxide (Merck, Darmstad, Germany). After evaporation of the solution to dryness, the resulting salt was dried by evaporation of 3 x 30 mL of anhydrous pyridine (Sigma). Then, the dry nucleoside was dissolved in a mixture of 50 mL of anhydride acetic (Sigma) and 175 mL of anhydrous pyridine. The solution was stirred in the dark for 16 h. After completion of the reaction, the excess of anhydride acetic was destroyed by the addition of 50 mL of ethanol. Then, di-O-Ac-dGuo (la)was purified, after lyophilization, by preparative liquid chromatography, using a mobile phase constituted of 40% of methanol in water, the flow rate being 100 m u m i n . The collected fraction (capacity factor: k’ = 4.0) gave 2.2 g of di-O-Ac-dGuo(la)(yield 60%). ‘H NMR (200.13 MHz, 2Hz0, TSP) 6 2.12 (s, 3H, CH3CO), 2.23 (s,3H, cH3co),3.06 (m, l H , H-2’),2.79 (m, l H , H-21, 4.42 (m, 2H, H-5’ H-51, 4.55 (m, l H , H-4‘1, 5.59 (m, l H , H-3’1, 6.40 (t, l H , H-l’), 8.07 (s, l H , H-8). NMR (50.3 MHz, (C2H3)2S0,TMS) 6 20.5 (cH3co),20.7 (CH&O), 35.6 (C-2’),63.7 (C-57, 74.6 (C-37, 81.6 (C-4‘), 82.8 (C-l’), 116.9 (C-5), 135.4 (C8), 151.2 (C-4), 153.8 (C-2), 157.0 (C-6), 169.9 (CHsCO), 170.1 (CH3CO). 4,8-Dihydro-4-hydroxy-8-oxoguanine (4-OH-8-oxoGua) was quantitatively obtained by chemical hydrolysis of the corresponding nucleosides (4-OH-8-oxodGuo)using hydrogen fluoride stabilized in pyridine (HFiPyr) as previously described by Polverelli et al. (43)with the following modifications. Typically, 1mg of 4-OH-8-oxodGuo (2 or 3) was treated with 50 pL of HF/ Pyr a t 37 “C during 30 min. Then, the residual HF was neutralized by addition of 1mL of an aqueous solution containing 80 mg of calcium carbonate (Sigma). A vigorous agitation was maintained until neutral pH was obtained. The resulting insoluble salts (CaF2) and the excess of calcium carbonate salts were eliminated by filtration through a Millex-GS 0.22 pm filter (Millipore). Then, pyridine was removed by repeated lyophilization, and the final residue was analyzed by HPLC. 4-OH-8oxoGua was separated by HPLC using an analytical NH2 silica gel column and a mixture of acetonitrile and 25 mM ammonium formate (80:20) as the mobile phase, the flow rate being 1 m u min. The collected fraction (k’ = 3.11, as detected by U V absorption a t 230 nm, was lyophilized twice in order to eliminate ammonium formate prior to 13C NMR and mass spectrometry analysis. FAB/MS: m / z (relative intensity) (positive mode) 206 (16, [M Na]+), 184 (100, [M + HI+). 13C NMR (50.3 MHz, (C’H3)2SO, TMS) 6 78.9 (C-4), 156.2 (C-8), 170.8, 172.1, 181.5. Photosensitization of 2-Deoxyguanosine. One hundred milliliters of an aqueous (either water Milli-Q, pH 6, or 2Hz0
+
Oxidation of DNA Induced by Singlet Oxygen
Chem. Res. Toxicol., Vol. 8, No. 3, 1995 381 Table 1.400.13MHz lH NMR Features of 2-Deoxyguanosine (1) and the Main Singlet Oxygen Oxidation Product 2.3.2a. and 3a in 2He0
384
100%
protons 80%
$
60%
m U C 3
??
2
3
5.87 2.55 2.30 4.34 3.94 3.70 3.68
5.90 2.34 2.21 4.42 3.98 3.74 3.69
7.4 6.4 -14.1 6.1 3.6 2.6 3.7 4.7 -12.6
184
n m
.-9 1
1 6.30 2.79 2.52 4.64 4.14 3.82 3.77 7.99
40%
405 20%
7.6 6.7 -13.9 6.6 4.0 3.9 4.9 6.4 -12.0
2a
3a
5.90 2.60 2.40 5.23 4.28 4.18 4.15
5.90 2.41 2.32 5.30 4.29 4.26 4.28
2.18, 2 .20 2.18, 2.19 8.8 9.2 5.9 5.8 -14.2 -14.0 6.5 5.8 2.4 2.2 2.4 2.0 3.8 3.5 6.2 6.1 -11.7 -12.7
8.7 6.3 -13.8 6.2 3.1 3.0 4.8 6.0 -12.0
Table 2. Chemical Shifts of the Exchangeable Protons of 3',5-Di-O-acetyl-2-deoxyguanosine (la) and Photoproducts 2a and 3a in (C2H&S0
0% 100
0
200
300
500
400
m/z Figure 1. FABMS (positive ion mode) spectrum of photooxidation product 2a. purchased from Merck) 1 mM solution of either dGuo (1)or its corresponding di-0-acetylated derivative la, containing 0.5 mM methylene blue, was irradiated for 1 h at room temperature in a large flask (15 cm diameter). Photosensitization with the zinc complexed phthalocyanine was performed using sufficient sensitizer to give an optical density absorbance of 2 at the maximum absorption of the dye (44). After irradiation, the solution was evaporated to dryness, and the resulting residue was dissolved in 5 mL of H2O. Then, the solution was centrifuged a t 12000g in order t o eliminate the excess of the starting unmodified nucleoside. The supernatant was evaporated to dryness, and the residue was dissolved in a minimum of HPLC buffer prior to the injection onto the NH2 semipreparative HPLC column. Nucleoside Photooxidation Products. (4R*)-4,8-Dihy-
protons
la
2a
3a
1' 2' 2" 3' 4' 5' 5" 8 CH3 CH3 NH C2-NHz C4-OH
6.24 2.56 2.03 5.40 nd" nd nd 8.03 2.14 2.18 10.87 6.65
5.08 2.75 2.09 5.12 4.15 nd nd
5.46 2.30 1.97 5.05 nd nd nd
2.03 2.03 11.57 8.59 8.26
2.03 2.03 11.51 8.50 8.20
a nd = not determined due to the overlapping between signals of protons 4', 5', and 5".
Table 3.50.3 MHz lSC NMR Chemical Shifts of 2-Deoxyguanosine (1) and of the Main Singlet Oxygen Oxidation Products 2.3, 2a, 3a, and 4 in (C2H&S0
~~____________
carbons 1'
dro-4-hydroxy-8-oxo-3',5'-di-O-acetyl-2-deoxyguanosine2' 3' (Sa). The separation of di-0-Ac-dGuophotoproducts was achieved 4'
on a NH2 silica gel column under isocratic conditions using a mixture of acetonitrile and 25 mM ammonium formate (85:15) as previously described by Ravanat et al. (41).Under these conditions, the unmodified nucleoside l a was found to be eluted more rapidly (k' = 1.5) than the main type I (k' = 2.0) and the two predominant type I1 (K' = 3.1, 12' = 3.3) photooxidation products. Lyophilization of the combined fractions (K' = 3.1) gave 3.8 mg (yield 10%) of oxidation product 2a. The FABMS spectrum (positive ion mode) is shown in Figure 1. FABMS: m / z (relative intensity) (positive mode) 405 (25, [M Na]+), 384 (100, [M Nal+), 184 (50, [B + HI+). The 400.13 MHz IH NMR spectroscopic parameters, including chemical shifts and coupling constants of 2a in 2Hz0, are listed in Table 1. The chemical shifts of 2a in (C2H3)2S0are listed in Table 2. The 50.3 MHz NMR chemical shifts of 2a in (C2H3)2S0are listed in Table 3.
5' 4 8
m3co
1 83.2 40.0 71.2 88.0 62.1 151.4 136.1 117.0 (5) 157.5 (6) 154.1 (2)
2 82.6 36.3 70.8 86.4 62.0 80.2 156.0 169.9 172.2 180.3
3 81.1 35.0 70.6 86.0 62.1 79.2 155.7 169.3 171.7 180.8
CH3CO
2a 80.3 33.7 74.0 82.7 63.6 80.5 155.3 169.2 172.3 180.0 20.6 170.2
3a 79.6 32.6 74.0 81.3 63.8 79.5 156.0 169.5 171.7 180.5 20.6 170.2
+
4
78.9 156.2 170.8 172.1 181.5
reported in Tables 1 and 2, respectively. The 50.3 MHz 13C NMR chemical shifts of 3a are listed in Table 3. ( 4 R * ) . 4 , & D i h y ~ h y d r o x y - & o x o - 2 ' - d e o x y g (2). The main photooxidation products of dGuo (1)were separated on a NH2 semipreparative HPLC column under isocratic condition using a mixture of acetonitrile and 25 mM ammonium formate (80:20) a t a flow rate of 2.0 m u m i n . Under these ~4S*)-4,8-Dihydro-4-hydroxy-8-oxo-3',5'-di-O-acetyl-2conditions, it should be noted that methylene blue was found deoxyguanosine (3a). Lyophilization of the combined fracto be eluted in the void volume of the column ( 4 1 ) . The oxidized tions (k' = 3.3) gave 3.8 mg of 3a (yield 10%). The FABMS nucleosides were eluted in the same order as the corresponding spectrum of the compound was found to be similar to that of 3',5'-di-O-acetylated derivatives. Lyophilization of the combined photoproduct 2a. FABMS: m / z (relative intensity) (positive fractions (k' = 4.0) gave 3 mg of 2 (yield 10%). FABMS: m / z mode) 405 (25, [M Nal+), 384 (100, [M Nal+), 184 (50, [B (relative intensity) (positive mode) 344 (10, [M 2Nal+), 322 (25, [M Nal+), 300 (12, [M HI+). The 400.13 MHz lH NMR HI+). The 400.13 MHz 'H NMR spectroscopic parameters, including chemical shifts of 3a in 2H20 and (C2H3)2S0, are spectroscopic parameters including chemical shifts and coupling
+
+
+
+
+
+
+
382 Chem. Res. Toxicol., Vol. 8, No. 3, 1995
1
0.5
0 220
260
240
280
300
320
Figure 2. Ultraviolet absorption spectra of dGuo (1) (dashed line) and photooxidation product 2 (solid line).
220 L?-
240
260
280
300
320
340
Ravanat and Cadet was 2 m u m i n , and the detection was achieved by UV monitoring at 290 nm. The product contained i n the fraction (k’= 3.2) was purified twice on the HPLC system in order to remove the starting 2’-deoxyguanosine (1)that is eluted very closely (k’= 3.0) to 4. The product was characterized as 7,8-dihydro-8-oxo2’-deoxyguanosine (4) by comparison of its lH and 13C NMR spectroscopic features with those of the authentic sample previously described by Lin et al. (42). DNA Photosensitization. Calf thymus DNA (500 pg) (Boeringher, Mannheim, Germany), dialyzed against water during 30 min by using a VM 0.05 ,um Milipore filter, was dissolved in 5 mL of Milli-Q water that contained 50 pg of methylene blue. After stirring in the dark for 1h, the solution, saturated with oxygen by continuous air bubbling, was irradiated during 30 min in a 2.5 cm round-bottom glass flask. After irradiation, the DNA solution was reduced to about 200 pL in a Speed Vac concentrator (Savant Instrument, Farmingdale, NY). In order to eliminate the photosensitizer, the DNA was twice precipitated using 0.1 volume of 3 M (pH = 5.3) ammonium acetate and 2.5 volumes of ethanol. Then, the DNA pellet was dried by lyophilization prior to acidic hydrolysis. HFPyr Hydrolysis of DNA. Photosensitized DNA (500pg) was dissolved i n 50 pL of H F P y r . In a subsequent step, the hydrolysis was performed during 30 min at 37 “C. Then, the residual H F was neutralized by addition of 1mL of a n aqueous solution containing 80 mg of calcium carbonate. A vigorous agitation was maintained until neutral pH was obtained. The resulting insoluble salts (CaF2) and the excess of calcium carbonate salts were eliminated by filtration through a MillexGS 0.22 pm filter. Then, pyridine was removed by repeated lyophilization, and the final residue was dissolved in 100 pL of water prior to HPLC analysis for the detection of the oxidation products of guanine. Ten microliters of the latter solution was used for the detection of 8-oxoGua by amperometry (40). Detection of 4-OH-8-oxoGua was performed i n two steps. The residual 90 pL was injected onto a CIS reverse phase column which was subsequently eluted by using 10 mM ammonium formate as the mobile phase in order to eliminate the unmodified nucleobases. 4-OH-8-oxoGua which was rapidly eluted was collected in a large fraction, k’ < 1, with a recovery of almost 100%. After repeated lyophilization, the content of the collected fraction was analyzed by HPLC under isocratic conditions on a n analytical NH2 column with a mobile phase constituted of a mixture of acetonitrile and 25 mM ammonium formate (80:20), the flow rate being 1 mlimin. Quantitation was achieved by UV measurement a t 230 nm using the synthetic 4-OH-8-oxoGua (k’ = 3.1) as the external standard.
Results
Isolation of the Singlet Oxygen Oxidation Photoproducts of 2’-Deoxyguanosine. The separation of the methylene blue mediated photooxidation products of constants of 4-OH-8-oxodGuo (2) obtained in 2H20 are listed in dGuo (1) was achieved on a silica gel NH2 column as Table 1. The 50.3 MHz 13CNMR chemical shifts of 2 in (C2H3)2previously described by Ravanat et al. (41). The methSO are reported in Table 3. The UV absorption spectrum of ylene blue photosensitization reaction of 1 was found to the 4R* diastereomer of 4-OH-8-oxodGuo (2) is shown in Figure give rise to the formation of four main stable photoprod2. The spectrum exhibits a shoulder at 230 nm; the molecular ucts. Under the HPLC analytical conditions, the 4R* and absorption coefficient at this wavelength was determined to be 4S* diastereomers of 4-OH-8-oxodGuo (2 and 3) are well 5240 mol-I cm-I L-l for both photoproducts 2 and 3. The separated from the starting 2’-deoxyguanosine (1) and circular dichroism spectrum of 2 is shown i n Figure 3. 2,2-diamino-4-[(2-deo~-~-~-erythro-pen~fix-anosyl)arnino](BS*)-4,B.Dihydro-4-hydroxy.&oxo-2’-d~~~os~e (3). Lyophilization of the combined fractions (k’= 4.5) gave 3 mg of 5-(2H)-oxazolone (5), one of the main stable type I homogeneous 3 (yield 10%). FABNS: m l z (relative intensity) photooxidation products (411. The other type I photo(positive mode) 344 (10, [M + 2Nal+), 322 (25, [M + Nal+), 300 product, (2S)-2,5’-anhydr0-1-(2-deoxy-P-~-erythro-pento(12, [M + HI+). The 400.13 MHz lH NMR spectroscopic furanosyl)-5-guanidinylidene-2-hydroxy-4-oxoimidazoliparameters and the 50.3 MHz 13C NMR chemical shifts are dine (6) (45), coelutes with starting 2‘-deoxyguanosine listed i n Tables 1 and 3, respectively. The circular dichroism under these conditions. The formation of 5, whose spectrum of 3 is shown in Figure 3. characterization is reported elsewhere (46),involves the 7,8-Dihydro-8-oxo-2-deoxyguanosine (4). 7,8-Dihydrotransient formation of a purine radical cation (46, 47). 8-oxo-2’-deoxyguanosine (4) was purified on a semipreparative The purity of each of the two diastereomers of 4-OH-8home-packed CISreverse phase column using a mixture (93:7) oxodGuo was greater than 95% as determined by ‘H of water and acetonitrile as the mobile phase. The flow rate
Figure 3. Circular dichroism spectra of photooxidation products 2 (solid line) and 3 (dashed line) i n H2O.
Chem. Res. Toxicol., Vol. 8, No. 3, 1995 383
Oxidation of DNA Induced by Singlet Oxygen
fl
4a 1 R = CH3CO
/
0
0
OR
Type II
NH,
____, IO2
OR
1
1 :R=H la:R=CH3CO
dR I OR
2,3 : R = H 2a, 3a : R = CU3CO
6R Figure 4. Mechanism of formation of the main type I1 oxidation products of dGuo (1) and 3’,5’-di-O-acetyl-2’-deoxyguanosine(la) in neutral aqueous solution.
NMR spectroscopy. It is important to note that the separation of the photoproducts of 3’,5’-di-O-acetyl-Ydeoxyguanosine (la) was achieved under similar chromatographic conditions by using a mixture of acetonitrile and ammonium formate (85:15). Again, three main products, l a , 2a, and 3a, were detected, corresponding to the 3’,5’-di-O-acetylated derivatives of the unprotected nucleosides 1,2, and 3, respectively. Methylene blue was found to have no interaction with the amino silica gel column and was eluted in the void volume of the HPLC column. In addition, it should be noted that another singlet oxygen oxidation product of 1 was found to be generated in a relatively lower yield as compared to the two diastereomers 2 and 3. This compound, which was characterized as 8-oxodGuo (41, is not separated from 1 under the analytical HPLC conditions used. Structure Assignment. The two main methylene blue photooxidation products (2 and 3) arising from type I1 photosensitization reaction (vide infra) of dGuo (1)as well as the corresponding 3’,5’-di-O-acetylated derivatives 2a and 3a were unambiguously assigned on the basis of FAB/MS mass spectrometry analysis and extensive ‘H and 13C NMR measurements (the structure of the 4R* and 4S* diastereomers of 3’,5’-di-O-acetyl-4,8-dihydro4-hydroxy-8-oxo-2’-deoxyguanosine is illustrated in Figure 4). Mass Spectrometry. The FAB/MS spectrum of photoproduct 2a (Figure 1)shows a notable pseudomolecular ion a t m l z 384 ([M HI+) together with a quasimolecular ion a t m l z 405 ([M Nal+). This is indicative of the gain in mass of 32 amu from the starting nucleoside la, corresponding to the incorporation of two oxygen atoms. In addition, the presence of a fragment a t m l z 184 ([B HI+) indicates that the incorporation had occurred within the base moiety. Additional structural
+
+
+
information was inferred from the mass spectra of the nonacetylated nucleosides 2 and 3. The mass spectrum of (2)(data not shown) shows a notable pseudomolecular ion a t m l z 299 together with a quasi-molecular ion at m l z 322 ([M Nal+). The fragment a t m l z 184 ([B HI+) is also detectable. These results are indicative of the molecular formula of C10H13N506for the nucleosides 2 and 3 and C5H&,03 for the corresponding base moiety. Further confirmation was provided by the FAB/MS spectra features of the corresponding base (4-OH-8oxoGua) obtained after acidic hydrolysis of 2 or 3 which exhibit a pseudomolecular ion and a quasi-molecular ion ([M Nal+) a t m l z 184 and 206, respectively (vide infra). ‘HNMR Spectroscopy. Inspection of the IH NMR data (Table 1) shows that the sugar moiety of the photooxidation products 2 and 3 and their di-0-acetylated derivatives 2a and 3a is intact. Important structural information is provided by the lack of the H8 signal, in the products 2 and 3 as well as 2a and 3a. This indicates that one of the two incorporated oxygen atoms is likely to be attached to the C8 carbon of the guanine moiety of the oxidation products. It should be added that the IH NMR features are similar for compounds 2 and 3 (and also for 2a and 3a). The relative high magnitude value for the trans coupling constant J11.y and the low value of the two other trans coupling constants J2tt-y and 5 3 ’ 4 suggest that the two nucleosides adopt preferentially a C2’ endo puckered conformation (48). In addition, the downfield shift of the NMR signal of the H2’ proton, together with the upfield shift of the C2’ signal, with respect to those of 1, is indicative of a preferential syn conformation (49). In addition, a significant destabilization of the gauche-gauche rotameric population may be inferred from the high value of the sum of J4f.v and J4f-5” coupling constants.
+
+
+
384 Chem. Res. Toxicol., Vol. 8, No. 3, 1995
Ravanat and Cadet
The lH NMR spectra of 2a and 3a in (C2H&S0 exhibit C8 three additional signals which may be easily chemically A exchanged by addition of DzO (Table 2). Two of them correspond to the resonance signal of the exocyclic amino group and a N(H) respectively. The third exchangeable signal in the NMR spectrum of 2a and 3a is likely to be assign to the proton of a hydroxyl group a t position 4. The exchangeable protons were also observed in the two corresponding nonacetylated photoproducts 2 and 3. 13C NMR Spectroscopy. The assignment of the osidic carbons (Table 3) was unambiguously achieved on I I I 1 the basis of heteronuclear decoupling experiments. The chemical shifts of the five osidic carbons of nucleosides 2 160 140 120 100 80 60 40 ppm and 3 and of the seven osidic carbons of the corresponding 3',5'-di-O-acetylated derivatives 2a and 3a are reported in Table 3. It should be noted that no important shift c4 was observed for the resonance signals by comparison with those of the osidic carbons of dGuo (1)with the exception of the C2' signal. Comparison of the chemical ca shifts of the base moiety of 2 and 3 with those of 1 indicates a notable upfield shift for the resonance signal of C4 carbon (Table 3). Such a large upfield shift indicates a change in the carbon hybridization from a sp2 to a sp3 structure, due to the addition of a n oxygen molecule to the base moiety. Long range scalar correlations between anomeric proton and carbons of the base moiety were used in order to assign the C4 and C8 carbons. The related long range heteronuclear scalar correlations of photoproducts 2 and dGuo (1) are shown in Figure 5. A correlation between the anomeric proton and two carbons of the purine ring I I I 1 I 1 I 180 160 140 120 100 80 60 40 of 1 was observed (Figure 5, part A). These carbons were ppm found to be the C8 and C4 carbons in agreement with Figure 6. Long range scalar correlations between the anomeric available data from the literature (50,511. This indicates proton and C4 and C8 carbons of the purine moiety of 1 (part that a three bond scalar correlation between H1' and the A) and 2 (part B), respectively. The bottom of each part C4 and C8 guanine carbons is present. Similar correlarepresents the I3C NMR spectrum of 1(part A) and 2 (part B), and the top shows the long range correlation between the tions were observed for photoproduct 2 (Figure 5, part anomeric proton and the carbons of the corresponding nucleoB). Therefore, these observations allowed us to assign side. the C4 and C8 carbons of 2 (Table 3). A similar result was obtained for the other diastereomer 3, providing acetylated derivatives 2a and 3a) was carried out by confirmation that the hydroxyl group is attached to the using hydrogen fluoride stabilized in pyridine (HFPyr). C4 carbon in both photoproducts 2 and 3. This allowed the quantitative release of the two 4R* and W Absorption Spectrum. The UV absorption spec4s" enantiomers of 4-OH-8-oxoGuawhich were purified tra of 1 and 2 in H20 are shown in Figure 2. Confirmaby HPLC separation. tion of the sp2to sp3 change in the hybridation of the C4 FAB Mass Spectrometry. The FAB/MS spectrum of carbon upon addition of ' 0 2 is provided by the decrease 4-OH-8-oxoGua obtained in the positive mode shows a in the aromaticity of the nucleoside with the loss of pseudomolecular ion a t m l z 184 [M HI+ and a quasiabsorption around 260 nm. It is interesting to note that molecular ion a t m l z 206 [M Nal+. This could be the UV absorption spectrum of the photooxidation prodrationalized in terms of a molecular weight of 183, uct 2 exhibits a shoulder around 230 nm. The molecular corresponding to the elemental composition of C5HSN503, absorption coefficient at this wavelength was determined confirming the presence of two additional atoms of oxygen experimentally to be 5240 mol-l cm-l L-l. It should be within the guanine moiety. added that the other diastereomeric photoproduct 3, as 13C NMR. The 13C NMR features of 4-OH-8-oxoGua well as the corresponding 3',5'-di-O-acetylated derivatives (Table 3) indicate the presence of 5 carbon resonances, 2a and 3a, exhibits similar UV absorption features. as expected from the results obtained with the related Circular Dichroism. The two nucleosides 2 and 3 nucleosides 2a, 3a, 2, and 3. The chemical shift values exhibit quite similar but opposite n-n* transitions with are similar to those of the base moiety of the nucleosides two maxima a t 235 and 260 nm, respectively (Figure 3). 2 and 3. Therefore, the downfield shift signal of 4-OHThis strongly suggests a diastereomeric relationship 8-oxoGua (78.9 ppm) and the signal a t 156.2 ppm were between 2 and 3. assigned to the C4 and C8 carbons, respectively, by Altogether, the above spectroscopic data may be rationalized in terms of a 4,8-dihydro-4-hydroxy-8-oxoguanine comparison with the data obtained for 2 and 3 (Table 3). structure for the aglycon of 2 and 3 as well as for 2a and Taken together, all the spectrometric data can be 3a. rationalized in terms of a 4,8-dihydro-4-hydroxy-8-0~0Characterization of 4,8-Dihydro-4-hydroxyy-8-ox-2'-deoxyguanosine structure for photoproducts 2 and 3 oguanine (COH-8-oxoGua). Chemical hydrolysis of (Figure 4) which are the two 4R* and 4S* diastereomers. the two photoproducts 2 and 3 (or the corresponding The photoproducts 2a and 3a correspond to the 3',5'-di-
I1
l
l
I
I
l
l
I
l
l
l
I
I
l
lB
I
+
+
Oxidation of DNA Induced by Singlet Oxygen
Chem. Res. Toxicol., Vol. 8, No. 3, 1995 385
Table 4. Relative Abundance of the Major Ions Observed in the FABMS Spectra (Positive Ion Mode) of Photoproduct 2a (MW = 383) and 8-OxodGuo (4) (MW = 283) Obtained after Methylene Blue Photosensitization in the Presence of lSOz 2a
[M [M
+ HI+
+ Nal+
8-0~0dGuo (4) [M HI+
+ [B+ HI+
mlz
re1 abundance
384 386 388 406 408 410
75 100 40 50 68 26
284 286 170 168
45 100 80 40
'
0
0
10
20
30
40
50
60
Time (min)
4-OHd-oxodGuo
+
+
0.4
0.2
+
+
0.8
0.6
+
+
1.2
g
0-acetylated derivatives of 2 and 3, respectively, whereas product 4 may be assigned a s 4,8-dihydro-4-hydroxy-8oxoguanine. Mechanism of Formation. Oxygen Incorporation. In order to establish the origin of the two additional oxygen atoms in the main photooxidation products 2a and 3a, di-0-Ac-dGuo (la) was exposed to photoexcited methylene blue in a n aqueous solution enriched with l80 isotopically labeled oxygen. The incorporation of l80in 2a was determined by FABI'MS (positive ion mode) analysis (Table 4) after HPLC separation of each of the two diastereomers. The presence of the pseudomolecular ion [M HI+ a t m / z = 388 and m I z = 386, as well as the ion [M Nal+ a t m l z = 410 and m / z = 408, indicates that the two oxygen atoms incorporated in the photoproducts come from molecular oxygen and not from water. It should be noted that the presence of the pseudomolecular ion [M HI+ at mlz = 386 and [M Nal+ a t m / z = 406, respectively, indicates that the enrichment of the irradiated solution in 1 8 0 2 was not quantitative but approximately 60%, due to air contamination. In this respect, it is interesting to note that a similar yield of incorporation of l80was observed for the main stable type I photooxidation product (46). A similar experiment was carried out in order to determine the origin of the additional oxygen atom in 8-oxodGuo (41, the other ' 0 2 oxidation product of 1. The FAEVMS (positive ion mode) spectrum of 4 obtained under these conditions showed the presence of a pseudomolecular ions [M HI+ a t m l z = 286 and a fragment [B HI+ a t mlz = 170 (Table 4). This indicates that the C8 oxygen atom in 4 comes from molecular oxygen. Singlet Oxygen Participation. The methylene blue photosensitized oxidation of dGuo in aqueous aerated solution may proceed either through a radical (type I) and/or a singlet oxygen (type 11) mediated mechanism (8). In order to establish the origin of the main photoproducts, different experimental approaches were used: (a) Enhancing Isotopic Effect of 2H20.The enhancing effect of 2H20 was used in order to establish the participation of singlet oxygen in the mechanism of formation of photoproducts 2, 3, and 4. The formation of 8-oxodGuo (4) and 4-OH-8-oxodGuo (2 3) with respect to increasing periods of irradiation in HzO and 2H20 solution is illustrated in Figure 6. Results indicate that the formation of 4-OH-8-oxodGuois linear with the time of exposure of 1 to visible light. In addition, a n enhancing effect was observed when the irradiation was carried out in heavy water (Figure 6, lower part). This confirms the participation of singlet oxygen in the mech-
+
,
8oxodGuo
1
l2
0
1
2
4
3
5
Time (min)
-+- Hp
--t
*HP
Figure 6. Formation of photooxidation products 4-OH-8oxodGuo (sum of 2 and 3) (lower part) and 8-oxodGuo (4) (upper part) with respect to increasing periods of methylene blue mediated photosensitization of dGuo (1)in HzO (dashed line) or 2Hz0 (solid line).
anism of formation of such photoproducts. In contrast, no heavy water enhancing effect was observed for the formation of 5, whose formation involves a type I mechanism (46). It has to be noted that the formation of 4 is not linear with the time of irradiation (Figure 6, upper part); a plateau is reached a t approximately 1%of the original amount of dGuo. The plateau is reached more rapidly in 2H20 than in H2O solution, indicating that singlet oxygen is implicated in the mechanism of formation of 4. As we have already reported previously (44), a steady-state level in the production of 4 is obtained as its formation is balanced by a degradation reaction. This may be explained by the fact that 8-oxodGuo ( 4 ) is a better substrate for singlet oxygen than dGuo (1). Moreover, phthalocyanine, a photosensitizer that predominantly produces type I1 (44)mediated photosensitization reaction of 4, was found t o generate 4-OH-8-oxodGuo (2 and 3) as the main lo2 oxidation products, as inferred from HPLC and lH NMR analysis of the irradiated solution. (b) The Use of Selected Sensitizers. The use of selected sensitizers, such as riboflavin and benzophenone, which predominantly act through a type I photosensitization reaction, was found to generate 5 as the main photooxidation product. In contrast, a relatively low level, when detectable, of photoproducts 2, 3, and 4 (4OH-8-oxodGuo and 8-oxodGuo) was observed. On the other hand, the use of phthalocyanines and naphthalocyanines, well-known type 11photosensitizers, was found to give rise t o the predominant formation of the nucleosides 2, 3, and 4 with a very low yield of 5 (44).
386 Chem. Res. Toxicol.,Vol. 8, No. 3, 1995 3.0
2.0 2.5
+
1
4-OH-8oxodGuo
-
+-
Ravanat and Cadet of the guanine moiety of 1,is formed a t a very low level (less than 1%).
8oxodGuo
1
Discussion
1
0
2
3 Time (min)
4
5
Figure 7. Formation of photooxidation products 4-OH-8oxodGuo (2,3) (dashed line) and 8-oxodGuo (4) (solid line) versus increasing period of methylene blue mediated photosensitization of dGuo (1). Table 5. Yield" of Type I1 Photooxidation Products in Methylene Blue Mediated Photosensitization of Double-StrandedCalf Thymus DNA in Aqueous Aerated Solution control photosensitized
2 (10) 1500 (5700)
